Methods for preventing strokes by inducing tolerance to e-selectin

ABSTRACT

The present invention provides a method for reducing stroke-related tissue damage by treating a mammal with E-selectin. Preferably, this treatment induces E-selectin tolerance in the mammal. Another aspect of the invention is a method for inducing E-selectin tolerance in a mammal through intranasal administration of E-selectin, preferably including booster administrations. The present methods are especially adapted for use in patients at increased risk of stroke or who may become at increased risk of stroke.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage application under 35 U.S.C. 371 ofPCT/US01/16583, filed May 23, 2001, which claimed priority under 35U.S.C. 119 of U.S. Provisional Patent Application No.: 60/206,693, filedMay 24, 2000.

FIELD OF THE INVENTION

The present invention relates to methods for treating or preventingstrokes and methods for inducing tolerance to E-selectin. The presentmethods are especially adapted for use in patients at increased risk ofstroke or who may become at increase risk of stroke.

BACKGROUND

E-selectin (also known as ELAM-1, CD62, and CD62E) is a cytokineinducible cell surface glycoprotein cell adhesion molecule that is foundexclusively on endothelial cells. E-selectin mediates the adhesion ofvarious leukocytes, including neutrophils, monocytes, eosinophils,natural killer (NK) cells, and a subset of T cells, to activatedendothelium (Bevilacqua, et al., “Endothelial leukocyte adhesionmolecule 1: an inducible receptor for neutrophils related to complementregulatory proteins and lectins,” Science 243; 1160 (1989); Graber, etal., “T cells bind to cytokine-activated endothelial cells via a novel,inducible sialoglycoprotein and endothelial leukocyte adhesionmolecule-1” J. Immunol. 145: 819 (1990); Carlos, et al., “Humanmonocytes bind to two cytokine-induced adhesive ligands on culturedhuman endothelial cells: endothelial-leukocyte adhesion molecule-1 andvascular cell adhesion molecule-1” Blood 77: 2266 (1991); Hakkert, etal., “Neutrophil and monocyte adherence to and migration acrossmonolayers of cytokine-activated endothelial cells: the contribution ofCD18, ELAM-1, and VLA-4” Blood 78: 2721 (1991); and Picker, et al.,“ELAM-1 is an adhesion molecule for skin-homing T cells” Nature 349: 796(1991)).

The expression of E-selectin is induced on human endothelium in responseto the cytokines IL-1 and TNF, as well as bacterial lipopolysaccharide(LPS), through transcriptional upregulation (Montgomery, et al.,“Activation of endothelial-leukocyte adhesion molecule 1 (ELAM-1) genetranscription” Proc. Natl. Acad. Sci. 88: 6523 (1991)). E-selectin isexpressed in vascular endothelial tissue where cells have beenactivated. Pober, J. S., et al., “Two distinct monokines, interleukin 1and tumor necrosis factor, each independently induce biosynthesis andtransient expression of the same antigen on the surface of culturedhuman vascular endothelial cells,” J. Immunol. 136: 1680 (1986);Bevilacqua M. P., et al., “Identification of an inducibleendothelial-leukocyte adhesion molecule,” Proc. Natl. Acad. Sci. 84:9238 (1987). Activation of vascular endothelial cells is believed, in atleast some cases, to be involved in inflammatory vascular tissue damageleading to thrombosis (Fareed, J. et al., “Molecular markers ofhemostatic activation. Implications in the diagnosis of thrombosis,vascular, and cardiovascular disorders,” Clin. Lab. Med. 15: 39 (1995)).

It is well-established that vascular tissue damage and thrombosis areinvolved in the development of strokes. Decreased supply of oxygen andnutrients from the blood to brain cells due to vascular tissue damageand thrombosis leads to the death of brain cells, the clinicalmanifestations of a stroke, and causes the formation of detectablespaces left by these cells, called infarctions. Strokes are a majorcause of mortality in the world and account for tens of billions ofdollars of medical costs in the United States alone. Although sometreatments for stroke prevention are available, there is a need for moreeffective treatments that are applicable to a larger fraction ofafflicted patients.

Structurally, E-selectin belongs to a family of adhesion moleculestermed “selectins” that also includes P-selectin and L-selectin (seereviews in Lasky, “Selectins: interpreters of cell-specific carbohydrateinformation during inflammation” Science 258: 964 (1992) and Bevilacquaand Nelson, “Selectins” J. Clin. Invest. 91: 379 (1993)). Thesemolecules are characterized by common structural features such as anamino-terminal lectin-like domain, an epidermal growth factor (EGF)domain, and a discrete number of complement repeat modules(approximately 60 amino acids each) similar to those found in certaincomplement binding proteins.

Recently, new methods and pharmaceutical formulations have been foundthat induce tolerance, orally or mucosally (e.g., by intranasaladministration, using as tolerizers autoantigens, bystander antigens, ordisease-suppressive fragments or analogs of autoantigens or bystanderantigens). Such treatments are described in Wiener, H. et al.,“Bystander suppression of autoimmune diseases,” WO9316724 (1993);Brigham & Womens Hospital (US), “Enhancement of the down-regulation ofautoimmune diseases by oral administration of autoantigens,” WO9112816(1991); Weiner, H. et al., “Improved treatment of autoimmune diseases byaerosol administration of auto antigens,” WO9108760 (1991); Weiner, H.et al., “Methods of treating or preventing autoimmune uveoretinitis inmammals,” WO9101333 (1991); Weiner, H. et al., “Method of treating orpreventing type 1 diabetes by oral administration of insulin,” WO9206704(1992); Hafler, D. et al., “Bystander suppression ofretroviral-associated neurological disease,” WO940121 (1994); Weiner, H.et al., “Method of treating rheumatoid arthritis with type II collagen,”WO9407520 (1994); Weiner, H. et al., “Methods and compositions forsuppressing allograft rejection in mammals,” WO9207581 (1992);Wucherpfenning, K. et al., “Multiple sclerosis T-cell receptor,”WO9115225 (1991); Weiner, H. et al., “Suppression of proliferativeresponse and induction of tolerance with polymorphic class II mhcallopeptides,” WO9320842 (1993); Weiner, H. et al., “Suppression ofT-cell proliferation using peptide fragments of myelin basic protein,”WO9321222 (1993); and Weiner, H. et al., “Treatment of autoimmunediseases by oral administration of autoantigens,” WO9206708 (1992).

Intravenous administration of autoantigens (and fragments thereofcontaining immunodominant epitopic regions) has been found to induceimmune suppression through a mechanism called clonal anergy. Clonalanergy causes deactivation of only immune attack T-cells specific to aparticular antigen, the result being a significant reduction in theimmune response to this antigen. Thus, the autoimmune response-promotingT-cells specific to an autoantigen, once clonal anergized, no longerproliferate in response to that antigen. This reduction in proliferationalso reduces the immune reactions responsible for autoimmune diseasesymptoms (such as neural tissue damage that is observed in MS). There isalso evidence that oral administration of autoantigens (orimmunodominant fragments) in a single dose and in substantially largeramounts than those that trigger “active suppression” may also inducetolerance through clonal anergy (or clonal deletion).

A method of treatment has also been disclosed that proceeds by activesuppression. Active suppression functions via a different mechanism fromthat of clonal anergy. This method, discussed extensively in Weiner(1993), involves oral or mucosa administration of antigens specific tothe tissue under autoimmune attack. These so called “bystander antigens”cause regulatory (suppressor) T-cells to be induced in thegut-associated lymphoid tissue (GALT), or bronchial associated lymphoidtissue (BALT), or most generally, mucosa associated lymphoid tissue(MALT); MALT includes both GALT and BALT. These regulatory cells arereleased in the blood or lymphatic tissue and then migrate to the organor tissue afflicted by the autoimmune disease and suppress autoimmuneattack of the afflicted organ or tissue.

The T-cells elicited by the bystander antigen recognize at least oneantigenic determinant of the bystander antigen used to elicit them andare targeted to the locus of autoimmune attack where they mediate thelocal release of certain immunomodulatory factors and cytokines, such astransforming growth factor beta (TGF-β), interleukin-4 (IL-4), and/orinterleukin-10 (IL-10). Of these, TGF-β is an antigen-nonspecificimmunosuppressive factor in that it suppresses immune attack regardlessof the antigen that triggers the attack. (However, because oral ormucosa tolerization with a bystander antigen only causes the release ofTGF-β in the vicinity of autoimmune attack, no systemicimmunosuppression ensues.) IL-4 and IL-10 are also antigen-nonspecificimmunoregulatory cytokines. IL-4 in particular enhances Th2 response(i.e., acts on T-cell precursors and causes them to differentiatepreferentially into Th2 cells at the expense of Th1 responses). IL-4also indirectly inhibits Th1 exacerbation. IL-10 is a direct inhibitorof Th1 responses. After orally tolerizing mammals afflicted withautoimmune disease conditions with bystander antigens, increased levelsof TGF-β, IL-4, and IL-10 are observed at the locus of autoimmune attack(Chen, Y. et al., “Regulatory T cell clones induced by oral tolerance:suppression of autoimmune encephalomyelitis,” Science, 265: 1237-1240,(1994)). The bystander suppression mechanism has been confirmed by vonHerrath et al., “Oral insulin treatment suppresses virus-inducedantigen-specific destruction of beta cells and prevents autoimmunediabetes in transgenic mice,” J. Clin. Invest., 96: 1324-1331, (1996).

Although the induction of tolerance and a bystander effect has beendemonstrated for a number of antigens, there remains a need to developmethods for inducing tolerance to E-selectin, and a determination ofwhether such induction is possible. Furthermore, there remains a need todetermine whether E-selectin can be used as a bystander antigen for theinduction of tolerance that provides active suppression.

This invention meets these needs by providing a method for inducingtolerance of E-selectin. Furthermore, this invention provides a methodfor treating stroke by the treatment of E-selectin, apparently through abystander effect provided by E-selectin tolerance. These and otheradvantages, benefits, and uses of the present invention will be apparentto those of skill in the art upon a consideration of the presentspecification.

SUMMARY OF THE INVENTION

In general, the present invention relates to methods for preventingdamage to brain tissue resulting from blood vessel obstructions bytreatment with E-selectin. The present invention also relates to methodsfor inducing tolerization to E-selectin. More particularly, the presentinvention provides a method for reducing stroke-related tissue damage bytreating a mammal with E-selectin. Preferably, this treatment inducesE-selectin tolerance in the mammal. Another aspect of the invention is amethod for inducing E-selectin tolerance in a mammal through intranasaladministration of E-selectin, preferably including boosteradministrations.

This invention is especially useful in treatment of patients with aknown increased risk of stroke. Such patients would include, forexample, persons with high blood pressure (especially severe high bloodpressure or high blood pressure not controllable with conventional drugtreatment), persons with a family history of stroke, persons with one ormore previous strokes, diabetes, hypercholesterolemia, and the like. Thepresent method can also be used for treatment of patients scheduled toundergo drug treatments or surgical procedures that might increase therisk of stroke. The present methods can, however, be used withindividuals without known increased risk to stroke.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of cumulative infarct and hemorrhage areas (in mm²)over time (days). Shaded diamonds, ovalbumin 5 μg single regimen; opendiamonds, ovalbumin 5 μg regimen with boosters; shaded squares,E-selectin 5 μg single regimen; open squares E-selectin 5 μg regimenwith boosters.

FIG. 2 is a graph of cumulative number of infarcts and hemorrhages overtime (days). Shaded diamonds, ovalbumin 5 μg single regimen; opendiamonds, ovalbumin 5 μg regimen with boosters; shaded squares,E-selectin 5 μg single regimen; open squares, E-selectin 5 μg regimenwith boosters.

FIG. 3 is a graph of cumulative infarct area (in mm²) over time (days).Shaded diamonds, ovalbumin 5 μg single regimen; open diamonds, ovalbumin5 μg regimen with boosters; shaded squares, E-selectin 5 μg singleregimen; open squares, E-selectin 5 μg regimen with boosters.

FIG. 4 is a graph of cumulative number of infarcts over time (days).Shaded diamonds, ovalbumin 5 μg single regimen; open diamonds, ovalbumin5 μg regimen with boosters; shaded squares, E-selectin 5 μg singleregimen; open squares, E-selectin 5 μg regimen with boosters. Asterisksindicate data points where the values for ES groups are statisticallydecreased (p<0.0001) using a Cox Proportional Hazards Model over valuesobtained for control groups.

FIG. 5 is a graph of cumulative intraparenchymal hemorrhage areas (inmm²) over time (days). Shaded diamonds, ovalbumin 5 μg single regimen;open diamonds, ovalbumin 5 μg regimen with boosters; shaded squares,E-selectin 5 μg single regimen; open squares, E-selectin 5 μg regimenwith boosters.

FIG. 6 is a graph of cumulative number of intraparenchymal hemorrhagesover time (days). Shaded diamonds, ovalbumin 5 μg single regimen; opendiamonds, ovalbumin 5 μg regimen with boosters; shaded squares,E-selectin 5 μg single regimen; open squares, E-selectin 5 μg regimenwith boosters.

FIG. 7 is a series of bar graphs showing the effect of the tolerizingE-selectin regimen of the current invention on delayed-typehypersensitivity. For this experiment rats received an intranasaladministration of E-selectin, then were immunized with E-selectin in thefootpad prior to a booster immunization in the ear. The graphsillustrate the change in ear thickness of the ear that received thebooster immunization of E-selectin compared to the ear that did notreceive a booster administration. Panel A shows the increase inmillimeters in the ear with the booster, and panel B present theincrease in the ear receiving the booster in terms of percent change inear thickness. Graphs on the left side of each panel show data for ratsreceiving control PBS administrations. Graphs on the right side of eachpanel show data for receiving an E-selectin tolerizing regimen accordingto the current invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Methods for preventing or treating strokes with E-selectin. In oneaspect, the invention is a method for preventing strokes or reducingtissue damage caused by a stroke in a patient. The method comprisesadministering E-selectin to the patient. Preferably, E-selectin isadministered in a manner that induces tolerance, most preferablybystander-effect tolerance.

E-selectin (also known as ELAM-1, CD62, and CD62E) is a cytokineinducible cell surface glycoprotein cell adhesion molecule that is foundexclusively on endothelial cells. Structurally, E-selectin belongs to afamily of adhesion molecules termed “selecting” that also includesP-selectin and L-selectin (see reviews in Lasky, 1992 and Bevilacqua andNelson, 1993). These molecules are characterized by common structuralfeatures such as an amino-terminal lectin-like domain, an epidermalgrowth factor (EGF) domain, and a discrete number of complement repeatmodules (approximately 60 amino acids each) similar to those found incertain complement binding proteins.

Sources of E-selectin that can be used with the current inventioninclude E-selectin that has been substantially purified from naturalsources, recombinant E-selectin produced in procaryotic or preferablyeukaryotic host cells by methods well-known in the art, and fragments ofE-selectin. Furthermore, E-selectin can be replaced in the currentinvention by a small organic molecule or a small peptide with astructure that mimics that of a portion of, preferably an immunoreactiveportion of E-selectin is used. As used herein, “substantially pure”refers to a purity that allows for the effective use of E-selectin forthe treatment of stroke or induction of tolerance.

Preferably, E-selectin for the current invention is from the samespecies to which it is being administered. However, as illustrated inthe attached Examples, E-selectin is effective in at least someinstances in species other than that from which it originated. Forexample, human E-selectin is effective when administered to rats inaccordance with the methods of the current invention. Human E-selectinis comprised of 589 amino acids and has a molecular weight of 64 kDa.Nucleic acids encoding human E-selectin have been cloned and sequenced.(Bevilacqua, M. P. 1989). In one embodiment, the source of E-selectinused for the current invention is recombinant human E-selectin.

E-selectin tolerance induced by the bystander antigens of this inventionis dose-dependent over a broad range of dosages. However, there areminimum and maximum effective dosages which will, of course, varydepending on the method of administration. In other words, activesuppression of the clinical and histological changes accompanying orcausing a stroke occur within a specific dosage range, which, however,varies depending on the organism receiving the dosage, the route ofadministration, whether E-selectin is administered in conjunction withother co-stimulatory molecules, and the specific regimen of E-selectinadministration. For example, for oral administration E-selectin isgenerally administered in dosages ranging from about 0.005 and 500mg/day., more preferably about 0.05 to 50 mg/day. Preferred oral dosagesare from 0.5 ug to 50 mg per administration.

Many delivery routes are known in the art for inducing bystander-effecttolerance. These routes include mucosa routes such as, but not limitedto, enteral, oral, inhalable, and intranasal routes. For the currentinvention E-selectin tolerance is preferably induced by dropwise orspray application of E-selectin intranasally.

E-selectin formulations for use with the methods of the presentinvention may comprise inert constituents includingpharmaceutically-acceptable carriers, diluents, solubilizing agents,emulsifying agents, salts, and the like, as is well-known in the art.Preferred E-selectin formulations are intranasal formulations includingnormal saline solutions, such as, for example, isotonic andphysiologically buffered saline solutions and phosphate-buffered saline(PBS) solutions. The total volume of the intranasal formulations istypically less than 1 milliliter, preferably less than 100 μl. For oralor enteral E-selectin formulations for use with the present invention,tablets may be formulated in accordance with conventional proceduresemploying solid carriers well-known in the art. Capsules employed fororal formulations to be used with the methods of the present inventionmay be made from any pharmaceutically acceptable material, such asgelatin or cellulose derivatives. Sustained release oral deliverysystems and/or enteric coatings for orally administered dosage forms arealso contemplated, such as those described in U.S. Pat. No. 4,704,295,“Enteric Film-Coating Compositions,” issued Nov. 3, 1987; U.S. Pat. No.4,556,552, “Enteric Film-Coating Compositions,” issued Dec. 3, 1985;U.S. Pat. No. 4,309,404, “Sustained Release PharmaceuticalCompositions,” issued Jan. 5, 1982; and U.S. Pat. No. 4,309,406,“Sustained Release Pharmaceutical Compositions,” issued Jan. 5, 1982.

Examples of solid carriers include starch, sugar, bentonite, silica, andother commonly used carriers. Further non-limiting examples of carriersand diluents which may be used in the formulations of the presentinvention include saline, syrup, dextrose, and water.

E-selectin can also be administered in an aerosol or inhaled form.Examples of formulations for tolerizing agents administered byinhalation are provided in Weiner, H. et al., “Improved treatment ofautoimmune diseases by aerosol administration of auto antigens,”WO9108760 (1991). The antigens can be administered as dry powderparticles or as an atomized aqueous solution suspended in a carrier gas(e.g., air, N₂, and the like).

Dry aerosol in the form of finely divided solid particles of E-selectinthat are not dissolved or suspended in a liquid can also be used in thepractice of the present invention. E-selectin formulations may be in theform of dusting powders and comprise finely divided particles having anaverage particle size of between about 1 and 5 microns, preferablybetween 2 and 3 microns. Finely divided particles may be prepared bypulverization and screen filtration using techniques well known in theart. The particles may be administered by inhaling a predeterminedquantity of the finely divided or powdered material. The E-selectinformulations of the present invention may also be administered in theform of an aerosol spray using, for example, a nebulizer such as thosedescribed in U.S. Pat. No. 4,624,251 issued Nov. 25, 1986; U.S. Pat. No.3,703,173 issued Nov. 21, 1972; U.S. Pat. No. 3,561,444 issued Feb. 9,1971; and U.S. Pat. No. 4,635,627 issued Jan. 13, 1971. Other systems ofaerosol delivery, such as the pressurized metered dose inhaler (MDI) andthe dry powder inhaler (see, e.g., Newman, S. P. in Aerosols and theLung, Clarke, S. W. and Davia, D. eds. pp. 197-224, Butterworths,London, England, 1984) can be used when practicing the presentinvention.

One particularly useful animal model for the analysis of E-selectinformulations and their effectiveness in treating or preventing stroke isthe stroke-prone and spontaneously hypertensive SHR-SP rat (Okamoto, K.et al., “Establishment of the stroke-prone spontaneously hypertensiverat (SHR),” Circ. Res. (Suppl.) 34, 35: 1 (1974)). SHR-SP rats areavailable upon request from professor Yukio Yamori, Graduate School ofHuman and Environmental Studies, Kyoto University, YoshidaNihonmatsu-cho, Sakyo-ku, Kyoto, 606-8316, Japan. SHR-SP rats typicallydie of early-onset cardiovascular disease, sometimes as early as 14weeks of age, although some SHR-SP rats live to more than 56 weeks ofage. Frequently, the cardiovascular disease manifests as a stroke inthese rats. The occurrence of a stroke in these rats is diagnosed bymeasuring behavioral status that could be divided into 4 patterns: noabnormalities (grade 1), irritable (grade 2), lethargic (grade 3),akinetic (grade 4) (Yamori, U. et al., Japanese Criculation Journal 46:274 (1982)).

The brains of SHR-SP rats at the time of death, typically containnumerous infarcts and intraparenchymal hemorrage areas that can becounted and measured through microscopal analysis of brain sections. Theeffectiveness of an E-selectin formulation can be determined bycomparing infarct and intraparenchymal hemorrhage number and area ofSHR-SP rats that have been treated with a test E-selectin formulationadministered on a booster regime with those that have been treated withcontrol formulations consisting of only carrier components, non-specificantigens (e.g., ovalbumin), or E-selectin on a single tolerizationrather than a booster schedule. An example of this strategy is disclosedin the Examples section of this specification.

The optimum dosage of E-selectin is one generating the maximumbeneficial effect on brain tissue damage caused by thrombosis assessedas described above. An effective dosage causes at least a statisticallyor clinically significant attenuation of at least one marker, symptom,or histological evidence characteristic of stroke, such as thosedescribed above. Stabilization of symptoms or tissue damage, underconditions wherein control patients or animals experience a worsening ofsymptoms or tissue damage, is one indicator of efficacy of a suppressivetreatment.

Ascertaining the effective dosage range as well as the optimum amount ofE-selectin is determined using conventional methods and the teachings ofthe present application. For example, dosages for mammals and humandosages can be determined by beginning with a relatively low dose (e.g.,1 microgram) and progressively increasing it while measuring appropriateresponses (e.g., number of TGF-beta, IL-4, and/or IL-10 secreting cells;number and activation of immune attack T-cells in the blood (e.g., bylimiting dilution analysis and ability to proliferate); and/or diseaseseverity). The optimum dosage generates the maximum amount of preventionof strokes or the maximum protection from tissue damage in the braincaused by thrombosis while minimizing undesirable side effects.Potential side effects include the generation of pathogenicautoantibodies (Hu, W. et al., “Experimental mucosal induction ofuveitis with the 60-kDa heat shock protein-derived peptide 336-351,”Eur. J. Immunol. 28: 2444 (1998); Genain C. P., et al., “Latecomplications of immune deviation therapy in a nonhuman primate,”Science 274: 2054 (1996)) or a cytotoxic T lymphocyte response thatinduces autoimmunity (Blanas E., et al., “Induction of autoimmunediabetes by oral administration of autoantigen,” Science 274: 1707(1996)).

An effective dosage causes at least a statistically or clinicallysignificant attenuation of at least one manifestation of thrombosis inthe skull cavity such as, for example, the number or area of braininfarcts, the number or area of brain intraparenchymal hemorrhage, theoccurrence rate or time to onset of stroke, and the like. The maximumeffective dosage of a bystander antigen in humans can be ascertained bytesting progressively higher dosages clinical trials starting with arelatively low dosage, for example 0.5 μg per administration.

Preferred dosages for intranasal instillations are from 0.5 to 50 mg peradministration, preferably for humans approximately from 5 μg to 5 mgper administration. For rats, one preferred dosage is 5 μg peradministration. Preferred aerosol pharmaceutical formulations maycomprise, for example, a physiologically-acceptable buffered salinesolution containing between about 1 mg and about 300 mg of E-selectin.

Ascertaining the optimum regimen for administering E-selectin isdetermined in light of the information disclosed herein and well knowninformation concerning administration of bystander antigens andautoantigens. Routine variation of dosages, combinations, and durationof treatment is performed under circumstances wherein the effects ofsuch variations on the organism can be measured.

E-selectin is preferably used in the practice of this invention using aseries of administrations. Typically these administrations are spacedapart over a period of 1 to 2 weeks. For example and as further detailedin the Examples, E-selectin can be administered in five intranasaladministrations over a two week period. Preferably, this protocolinvolves administering E-selectin every other day for ten days.Preferably, the administration regimen is repeated in boosteradministrations which are generally administered several weeks apart. Inone preferred embodiment, booster administrations are given after everythree weeks. Booster administrations may include a series ofadministrations, as described above for initial administrations.

Cytokine and non-cytokine synergists can be used in conjunction withE-selectin in the present invention to enhance the effectiveness ofE-selectin tolerization. Administration “in conjunction with”encompasses simultaneous and sequenctial administration, as well asadministration in combined form or separately. Oral and parenteral useof cytokine synergists (Type I interferons) has been described inHafler, D. A. et al., “Treatment of autoimmune disease using oraltolerization and/or type 1 interferon,” WO9527499 (1995). Administrationof Th2 enhancing cytokines is described in Weiner H. L., et al.,“Treatment of autoimmune disease using oral tolerization and/orTh2-enhancing cytokines,” WO95275000(1995). For example, IL-4 and IL-10can be administered in the manner described in Weiner et al. Id.

Non-limiting examples of non-cytokine synergists for use in the presentinvention include bacterial lipopolysaccharides from a wide variety ofgram negative bacteria such as various subtypes of E. coli andSalmonella (LPS, Sigma Chemical Co., St. Louis, Mo.; Difco, Detroit,Mich.; BIOMOL Res. Labs., Plymouth, Pa.), Lipid A (Sigma Chemical Co.,St. Louis, Mo.; ICN Biochemicals, Cleveland, Ohio; Polysciences, Inc.,Warrington, Pa.); immunoregulatory lipoproteins, such as peptidescovalently linked to tripalmitoyl-S-glycarylcysteinyl-seryl-serine(P.sub.3 C55) which can be obtained as disclosed in Deres, K. et al.(Nature, 342: 561-564, “In vivo priming of virus-specific cytotoxic Tlymphocytes with synthetic lipopeptide vaccine,” 1989) or “Braun's”lipoprotein from E. coli which can be obtained as disclosed in Braun,V., Biochim. Biophys. Acta 435: 335-337, 1976; and cholera toxin β-chain(CTB) the synergist ability of which has been described (though not inconnection with abatement of autoimmune reaction) by Sun, J-B et al.,“Cholera toxin B subunit: an efficient transmucosal carrier-deliverysystem for induction of peripheral immunological tolerance,” Proc. Natl.Acad. Sci. (USA) 91: 10795 (1994). The effective dosage range fornoncytokine synergists for mammals is from about 15 ng to about 15 mgper kg weight and preferably 300 ng-12 mg per kg weight. The effectivedosage range for oral Type I interferon for mammals is from1,000-150,000 units with no maximum effective dosage having beendiscerned. Another active compound that may be useful in combinationwith E-selectin is methotrexate which is known to cause a marked Th2immune deviation with greatly increased IL-4 secretion when given on apulse regimen (Weiner et al., “Treatment of Autoimmune Disease UsingTolerization in Combination with Methotrexate,” U.S. Pat. No. 5,935,577(1999).

Ascertaining the optimum regimen for administering E-selectin and/or theco-stimulatory molecule is determined in light of the informationdisclosed herein and well known information concerning administration ofbystander antigens and autoantigens. Routine variation of dosages,combinations, and duration of treatment is performed under circumstanceswherein the effects of such variations on the organism can be measured.The co-stimulatory agent is preferably administered within 24 hours ofadministration of E-selectin. More preferably, it is administered at thesame time as E-selectin. Most preferably, both are administered in acombined oral formulation.

Not to be limited by theory, this invention is based on the hypothesisthat activation of the luminal surface of endothelium in a vascularsegment by proinflammatory cytokines such as tumor necrosis factor-alphaand interluekin-1-beta is a prerequisite for the development ofthrombosis or the evolution of inflammatory vessel damage in thatsegment. The general approach involves exposing lymphocytes inbronchial-associated lymphoid tissue (BALT) and perhaps gut-associatedlymphoid tissue (GALT) to an adhesion molecule antigen to producetolerized lymphocytes. The antigen is instilled intranasally. Thetolerized lymphocytes undergo “immune deviation,” thereby synthesizingand releasing transforming growth factor-beta (TGFβ; a cytokine thatcauses paracrine “bystander suppression” of proinflammatory cytokineproduction) when the same antigen is encountered again. The antigen isE-selectin, an adhesion molecule that is only expressed on theendothelial surface in vascular segments that have become activated. Theessence of this approach is, therefore, to program autologouslymphocytes to become mobile monitors that provide continuoussurveillance of vessels. When they encounter E-selectin in an activatedsegment they bind to that segment and become stimulated to produce TGFβ.The TGFβ then suppresses production of proinflammatory cytokines,reduces endothelial thrombogenecity and minimizes vessel injury. After asingle antigen exposure, tolerance of lymphocytes lasts for a period ofweeks and long-term maintenance of the tolerant state requires repeatedbooster exposures to the antigen.

Furthermore, the current invention provides methods of reducing thelikelihood of a stroke by a mechanism that may include specificallyreducing intracranial hemorrhage. Although not wishing to be limited bytheory, this conclusion is based on the following considerations relatedto intracranial hemorrhage and endoglin gene polymorphisms. Mutations ofthe endoglin gene have been associated with intracranial hemorrhage inpatients (Alberts, M. J. et al., “Endoglin gene polymorphism as a riskfactor for sporadic intracerebral hemorrhage,” Ann. Neurol., 41: 683(1997)). Endoglin appears to bind TGF-β and subsequently plays a role invascular maintenance and development. Impariment of endoglin functionappears to diminish the response of the endothelium to TGF-β resultingin an increased tendency to hemorrhage. E-selectin tolerization alsoappears to increase the number of TGF-β positive lymphocytes and mayincrease the release of TGF-β in vessel segments that are becomingactivated, as described above. This would be predicted to reduce thelikelihood of hemorrhage in the presence of endoglin. This ispotentially relevant to the observed elimination of intracranialhemorrhage in the group that received E-selectin tolerization andbooster tolerization, as described in Examples section below.

In another aspect, the current invention provides a method formitigating brain tissue damage following a stroke by administeringE-selectin to a patient immediately after, or preferably beforeoccurrence of the stroke. Preferably, E-selectin is administered in amanner that induces tolerance, as described below, most preferablybystander-effect tolerance. Considerations regarding E-selectin sources,doses, delivery routes, formulations, and the like, are described abovefor methods of preventing a stroke. As shown in the attached Examples,not only does E-selectin administration significantly reduce the numberof infarcts formed in a stroke-prone rat model, the infarcts that formare significantly smaller in size than control infarcts. Therefore,tolerance to E-selectin appears to minimize brain tissue damage inanimals that have a stroke while they are in a state of E-selectintolerance.

Method for Inducing E-Selectin Tolerance. One aspect of the currentinvention is a method for inducing E-selectin tolerance in a host. Themethod comprises intranasal administration of E-selectin. In onepreferred embodiment the protocol consists of booster intranasaladministrations of E-selectin.

In one embodiment, E-selectin tolerance is induced by a five by twoadministration protocol of five intranasal administrations of E-selectinover a period of two weeks. In a most preferred embodiment, this five bytwo administration is repeated at least once. Most preferably, thisbooster regimen is repeated every three weeks for the life of theorganism.

Preferred dosages, E-selectin sources, formulations, dosage volumes,regimens, and methods for analyzing results aimed at optimizing theseconsiderations for intranasal instillations for inducing E-selectintolerance are similar to those described above for the use of E-selectinadministration in stroke prevention. For example, the preferred dosagesrange from 0.5 μg to 50 mg per administration, preferably for humansapproximately from 5 μg to 5 mg per administration. Optimization of thedosage necessary for immune suppression involves no more than routineexperimentation, given the guidelines disclosed herein.

The current aspect of the invention for inducing E-selectin tolerancehas many utilities. For example, it can be used in preventing andtreating strokes and other forms of vascular disease such as coronaryartery disease. Additionally, it can be used in treating disorders inwhich E-selectin has been determined, or may be determined, to play arole, such as, for example, lung injury, psoriasis, contact dermatitis,inflammatory bowel disease, arthritis, and the like. (See, e.g.,Washington R., et al., “Expression of immunologically relevantendothelial cell activation antigens on isolated central nervous systemmicrovessels from patients with multiple sclerosis,” Ann. Neurol. 35: 89(1994); Bevilacqua (1989); Bevilacqua and Nelson, “Selectins,” J. Clin.Invest. 91: 379 (1993); Koch, et al., “Immunolocalization of endothelialand leukocyte adhesion molecules in human rheumatoid and osteoarthriticsynovial tissues,” Lab Invest. 64: 313 (1991); Mulligan, et al., “Roleof endothelial-leukocyte adhesion molecule 1 (ELAM-1) inneutrophil-mediated lung injury in rats,” J. Clin. Invest. 88: 1396(1991); and Mulligan, et al., “Protective effects of oligosaccharides inP-selectin-dependent lung injury,” Nature 364: 149 (1993)).

Assessment of the effect of E-selectin formulations on an immuneresponse to E-selectin can be made, for example, by determiningdiminution in certain inflammation markers, such as the number ofactivated T-cell clones directed against activated vascular tissue.Immunological tolerance can be measured by a number of methods that arewell-known in the art. In one preferred embodiment, delayed typehypersensitivity (DTH) response can measured in animals by injectingE-selectin into, for example, the footpad of an organism to be analyzedand then administering a booster injection, for example into a footpador an ear, at a later time, typically more than 1 week later, mostpreferably 2 weeks later. DTH reactions can be measured after theelicitation injection as the increase in swelling at the site of theantigen rechallenge. Footpad or ear swelling can be measured at, forexample, 0, 24 and 48 hr after challenge.

Methods for analyzing effects of E-selectin tolerance on strokeoccurrence or stroke-related damage using stroke-prone rats is discussedabove and in the Examples section. This method is useful as an indirectmeasure of the effectiveness of a particular dosage, formulation, andprotocol on inducing E-selectin tolerance through intranasaladministration of E-selectin.

The various publications, issued patents, published patent applications,and the like cited in this present application are hereby incorporatedby reference. The following examples describe and illustrate the methodsand compositions of the invention. These examples are intended to bemerely illustrative of the present invention, and not limiting thereofin either scope or spirit. Those of skill in the art will readilyunderstand that variations of the materials used in, and the conditionsand processes of, the procedures described in these examples can beused.

EXAMPLE 1

Reduction of brain infarcts by administration of E-selectin. The effectsof administration of E-selectin on infarcts of the brains ofstroke-prone rats was analyzed. Male and female stroke-prone andspontaneously hypertensive (SHR-SP) 8-10 week-old rats were obtainedfrom the NIH colony. (Okamoto (1974)). At 11 weeks of age,E-selectin-(soluble human E-selectin: human E-selectin lectin, EGF, CR1,CR2 domains, myc peptide tail) ovalbumin or vehicle (PBS) wereadministered intranasally. Purified human E-selectin was obtained fromProtein Design Laboratories (Fremont, Calif.).

E-selectin and control preparations were administered in the followingmanner: SHR-SP rats were divided into three groups: (1) a saline (PBS)control group, (2) an E-selectin administration group (ES group), and(3) an ovalbumin (OVA) administration group (OVA group). In addition, ESand OVA groups were divided into single (non-booster) and repetitive(booster) administration groups. For the control group, 20 μl ofphosphate-buffered saline (PBS) was administered into each nostril everyother day for 10 days for a total of 5 administrations. For the ESnon-booster group, 2.5 μg E-selectin in 20 μl PBS was administered intoeach nostril every other day for 10 days for a total of 5administrations. For the ES booster group, an initial 2.5 μg ofE-selectin in 20 μl PBS was administered as above for the non-boostergroup; additionally, 2.5 μg of E-selectin in 20 μl of PBS wasadministered intranasally into each nostril every other day for 10 days(3 weeks after the first E-selectin course) and repeated every 3 weeksuntil the animals were sacrificed. For the OVA non-booster group, 2.5 μgovalbumin in 20 μl PBS was administered into each nostril every otherday for 10 days for a total of 5 administrations. For the OVA boostergroup, an initial 2.5 μg of ovalbumin in 20 μl PBS was administered intoeach nostril as above for the non-booster group; additionally, 2.5 μg ofovalbumin in 20 μl of PBS was administered intranasally into eachnostril every other day for 10 days (3 weeks after the first ovalbumincourse) and repeated every 3 weeks until the animals were sacrificed.

The rats were evaluated for physical and neurological signs of stroke.These evaluations included an assessment of excitement (i.e.,piloerection, hyperkinesis), hyperirratibility (i.e., jumping, trying toescape), behavioral and psychological depression (i.e., hypokinesis,hyposthenia, hypotenia, hyporesponsiveness), motion disturbance (i.e.,transient episode of repetitiv lifting of paws, ataxia, paresis,paralysis), and late symptoms observed near the time of death (i.e.,apathy, coma, urinary incontinence). The rats were also monitored bymeasuring arterial blood pressure, body weight, heart weight, andarterial blood gas using methods know in the art.

Infarcts were evaluated in the following manner. When animals showedsigns of cardiac failure, kidney failure, or stroke, they were perfusedand their brains were removed for histology and image processing.Sections from 8 predetermined stereotactic levels were cut from eachbrain (total of 240 sections). The number and area of infarcts orhemorrhages were determined for each section from each animal.Statistical significance of E-selectin administrations was determined bycomparing E-selectin groups to control groups by a Cox ProportionalHazards Model.

The animals lived for variable periods from 14 weeks to the terminationof the experiment at 56 weeks. Deaths were caused by heart failure andkidney failure secondary to severe hypertension (mean systolic bloodpressure 215 mm Hg), as well as by strokes. Average age at time of deathand average systolic blood pressure did not differ among theexperimental groups.

The experimental group of animals which received E-selectin maintainedwith booster administrations had a statistically significant reductionin the frequency and area of infarcts compared to control groups(p<0.0001) (FIGS. 1-7). Mean area of infarcts decreased from betweenabout 6.873 mm² to about 27.718 mm² in control and single administrationE-selectin groups to about 0.002 mm² in the E-selectin booster group(i.e., a greater than 99% reduction; see Tables I-IV, FIGS. 1 and 3).Mean number of infarcts decreased from about 3.0 to about 7.3 forcontrol and single administration E-selectin groups to about 0.3 inE-selectin booster groups (i.e., a greater than 91% reduction; seeTables I-IV, FIGS. 2, and 4). Intraparenchymal hemorrhages were absentfrom the E-selectin booster group, but were present at an average numberof from about 3.2 to about 2.3 per brain section analyzed in control andsingle E-selectin administration groups (see Tables I-IV and FIGS. 1, 2,5, and 6).

TABLE I Group OVA Data Intraparenchymal Sample Infarcts Hemorrhage (sex)Number Area (mm²) Number Area (mm²) 1 (female) 13 6.966 2 0.439 2(female) 0 0 0 0 3 (female) 1 0.062 15 0.390 4 (female) 19 133.850 40.950 5 (male)   15 70.559 1 0.021 6 (male)   10 10.308 0 0 7 (female) 00 0 0 8 (female) 0 0 0 0 Mean 7.3 27.718 2.8 0.225

TABLE II Group OVAb Data Intraparenchymal Sample Infarcts Hemorrhage(sex) Number Area (mm²) Number Area (mm²) 1 (female) 0 0 0 0 2 (male)  3 0.734 1 4.784 3 (female) 21 40.502 17 1.372 4 (female) 0 0 0 0 5(female) 0 0 1 0.063 6 (female) 0 0 0 0 Mean 4.0 6.873 3.2 1.037

TABLE III Group ES Data Intraparenchymal Sample Infarets Hemorrhage(sex) Number Area (mm²) Number Area (mm²) 1 (female) 0 0 0 0 2 (male)  0 0 0 0 3 (female) 9 13.488 5 0.177 4 (female) 14 77.909 13 7.553 5(female) 0 0 0 0 6 (female) 1 0.012 0 0 7 (male)   0 0 0 0 8 (male)   00 0 0 Mean 3.0 11.426 2.3 0.966

TABLE IV Group ESb Data Intraparenchymal Sample Infarcts Hemorrhage(sex) Number Area (mm²) Number Area (mm²) 1 (male)   0 0 0 0 2 (female)0 0 0 0 3 (female) 0 0 0 0 4 (female) 0 0 0 0 5 (male)   1 0.003 0 0 6(female) 0 0 0 0 7 (female) 1 0.011 0 0 8 (male)   0 0 0 0 Mean 0.30.002 0 0

EXAMPLE 2

Induction of tolerance to E-selectin. An analysis was performed todetermine whether tolerance to E-selectin was induced by the intranasaladministration protocol of E-selectin described above, which resulted indecreased stroke-related tissue damage. For this analysis, eitherE-selectin or control PBS preparations were administered to rats asdescribed in Example 1 for the non-booster groups. Delayed-typehypersensitivity (DTH) was analyzed by injecting 5 μg of E-selectin in50 μl of PBS and 50 μl of complete Freund's adjuvant into hindpads(s.q.) 14 days after intranasal administration. Fourteen days later, therats were rechallenged by injecting 5 μg E-selectin in 50 μl PBS intothe ear and ear thickness was measured with microcalipers (Mitsutoyo) 48hours later.

Results of the delayed-type hypersensitivity assay demonstrated thatintranasal instillation of human E-selectin induced tolerance.Administration of E-selectin intranasally prior to footpad injection andelicitation ear injection resulted in a significant suppression of earswelling compared to control groups (FIG. 7), as measured with Mitsutoyomicrocalipers. This data demonstrates that the E-selectin administrationprotocol used induced tolerance to E-selectin.

1. A method for reducing stroke-related tissue damage in a patient, saidmethod comprising a series of intranasal administrations of humanE-selectin.
 2. The method of claim 1, wherein the series of intranasaladministrations comprise a first series intranasal administrations ofhuman E-selectin and a booster administration of human E-selectincomprising a second series of intranasal administration of humanE-selectin.
 3. The method of claim 2, wherein the first series ofintranasal administrations and the second series of intranasaladministrations each comprise five administrations of human E-selectinover a period of two weeks.
 4. The method of claim 1, wherein eachE-selectin administration is in an amount effective to induce E-selectintolerance in the patient.
 5. The method of claim 1, wherein the humanE-selectin is soluble human E-selectin.
 6. The method of claim 1,wherein the human E-selectin comprises human E-selectin lectin, EGF,CR1, and CR2 domains.